Translation initiation has been demonstrated to be a key step in cell proliferation, differentiation and apoptosis. However, the mechanism(s) by which translation initiation is regulated remains largely unknown. We have demonstrated that the translational repressor 4E-BP1 (eIF4E-binding protein 1) is dephosphorylated and therefore activated in response to the immunosuppressant drug, rapamycin, and that 4E-BP2 (eIF4E-binding protein 2) is regulated during T cell maturation. We also demonstrated a differential regulation of 4E-BP1 and 4E-BP2 during myeloid differentiation, with an activation of 4E-BP1 specific to the monocytic/macrophage differentiation pathway and an upregulation of 4E-BP2 upon granulocytic differentiation. This proposal is intended to develop a better understanding of the process of translation initiation, focusing specifically on the role of 4E-BP proteins, in T lymphocytes and monocytes. The project takes advantage of mouse models with targeted disruption of either 4E-BP1 or 4E-BP2 and of technologies for transcriptomic and proteomic analysis. The specific aims are: (i) to identify genes that are differentially translated in response to rapamycin. We reported that rapamycin specifically inhibits eIF4E-dependent translation. In contrast, rapamycin increases internal initiation of translation, a mechanism independent of e1F4E activity and reported so far for only a few cellular mRNAs. Genes affected by rapamycin through translational control mechanisms, will be identified using an oligonucleotide array based approach for measuring total RNA and polysome-bound RNA levels and a proteomic approach for measuring changes in protein levels in response to rapamycin; (ii) to determine the role of 4E-BP1 in rapamycin signaling. For this purpose, we propose to investigate the effect of targeted disruption of the 4E-BP1 gene in a mouse model, on rapamycin signaling; (iii) to determine the role of 4E-BP2 in T cell maturation and apoptosis. We have reported that protein synthesis rates, eIF4E phosphorylation and 4E-BP2 expression are differentially regulated upon activation of human immature and mature thymocytes. We will investigate the effect of knocking-out 4E-BP2 gene in a mouse model, on T cell maturation and on their sensitivity to apoptosis; (iv) to determine the role of 4E-BP1 and 4E-BP2 in myeloid differentiation. We also reported specific regulation of 4E-BP1 and 4E-BP2 expression, during monocytic/macrophage or granulocytic differentiation pathways. We will identify genes whose translation may be regulated specifically or predominantly by 4E-BP1 or by 4E-BP2 in myeloid cells, using both oligonucleotide array based and proteomic approaches. These studies will provide novel and important insights into the contribution of 4E-BP1 and 4E-BP2 genes, to the changes in T cell and monocyte gene expression associated with their maturation, proliferation or apoptosis.